Penkala I, etal., Mol Cell Biol. 2016 May 31;36(12):1764-75. doi: 10.1128/MCB.01098-15. Print 2016 Jun 15.
Long noncoding RNAs (lncRNAs) can regulate gene expression in a cell-specific fashion during development. Here, we identify a novel lncRNA from the X chromosome that we named lncRHOXF1 and which is abundantly expressed in trophectoderm and primitive endoderm cells of human blastocyst-stage embryos.
lncRHOXF1 is a spliced and polyadenylated lncRNA about 1 kb in length that is found in both the nuclear and cytoplasmic compartments of in vitro differentiated human trophectoderm progenitor cells. Gain-of-function experiments in human embryonic stem cells, which normally lack lncRHOXF1 RNA, revealed that lncRHOXF1 reduced proliferation and favored cellular differentiation. lncRHOXF1 knockdown using small interfering RNAs (siRNAs) in human trophectoderm progenitors increased expression of viral response genes, including type I interferon. Sendai virus infection of human trophectoderm progenitor cells increased lncRHOXF1 RNA levels, and siRNA-mediated disruption of lncRHOXF1 during infection reduced the expression of viral response genes leading to higher virus replication. Thus, lncRHOXF1 RNA is the first example of a lncRNA that regulates the host response to viral infections in human placental progenitor cells, and we propose that it functions as a repressor of the viral response during early human development.
Penkowa M and Hidalgo J, Exp Neurol. 2001 Jul;170(1):1-14.
Experimental autoimmune encephalomyelitis (EAE) is an animal model for the human autoimmune disease multiple sclerosis (MS). Proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are considered important for induction and pathogenesis of EAE/MS disease, w
hich is characterized by significant inflammation and neuroglial damage. We have recently shown that the exogenous administration of the antioxidant protein zinc-metallothionein-II (Zn-MT-II) significantly decreased the clinical symptoms, mortality, and leukocyte infiltration of the CNS during EAE. However, it is not known how EAE progression is regulated nor how cytokine production and cell death can be reduced. We herewith demonstrate that treatment with Zn-MT-II significantly decreased the CNS expression of IL-6 and TNF-alpha during EAE. Zn-MT-II treatment could also significantly reduce apoptotic cell death of neurons and oligodendrocytes during EAE, as judged by using TUNEL and immunoreactivity for cytochrome c and caspases 1 and 3. In contrast, the number of apoptotic lymphocytes and macrophages was less affected by Zn-MT-II treatment. The Zn-MT-II-induced decrease in proinflammatory cytokines and apoptosis during EAE could contribute to the reported diminution of clinical symptoms and mortality in EAE-immunized rats receiving Zn-MT-II treatment. Our results demonstrate that MT-II reduces the CNS expression of proinflammatory cytokines and the number of apoptotic neurons during EAE in vivo and that MT-II might be a potentially useful factor for treatment of EAE/MS.
Poor outcome of extramedullary disease in multiple myeloma patients and lack of outcome predictors prompt continued search for new markers of the disease. In this report, we show circulating microRNA distinguishing multiple myeloma patients with extramedullary disease from myeloma patients without s
uch manifestation and from healthy donors. MicroRNA-130a was identified by TaqMan Low Density Arrays and verified by quantitative PCR on 144 serum samples (59 multiple myeloma, 55 myeloma with extramedullary disease, 30 healthy donors) in test and validation cohorts as being down-regulated in myeloma patients with extramedullary disease. Circulating microRNA-130a distinguished myeloma patients with extramedullary disease from healthy donors with specificity of 90.0% and sensitivity of 77.1%, patients with extramedullary disease from newly diagnosed multiple myeloma patients with specificity of 77.1% and sensitivity of 34.3% in the test cohort and with specificity of 91.7% and sensitivity of 30.0% in the validation cohort of patients. Circulating microRNA-130a in patients with extramedullary myeloma was associated with bone marrow plasma cells infiltration. Further, microRNA-130a was decreased in bone marrow plasma cells obtained from patients with extramedullary myeloma in comparison to bone marrow plasma cells of myeloma patients without such manifestation, but it was increased in tumor site plasma cells of patients with extramedullary disease compared to bone marrow plasma cells of such patients (p<0.0001). Together, our data suggest connection between lower level of microRNA-130a and extramedullary disease and prompt further work to evaluate this miRNA as a marker of extramedullary disease in multiple myeloma.
Platelets regulate cell-cell interactions and local circulation through eicosanoids from arachidonic acid. Sigma non-opioid intracellular receptor 1 (sigma-1 receptor) expressed in platelets and endothelial cells can regulate intracellular signalization. Our aim was to examine the influence of sub-c
hronic, in vivo-administered sigma-1 receptor ligands 2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate (PRE-084); N-benzyl-2-[(1S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]ethan-1-amine; dihydrochloride, a new compound ((S)-L1); and N-[2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethyl]-N-propylpropan-1-amine (NE-100) on the ex vivo arachidonic acid metabolism of the platelets and aorta of male rats. The serum level of sigma-1 receptor ligands was determined by liquid chromatography-mass spectrometry. Sigma-1 receptor and cyclooxygenase gene expression in the platelets were determined by a reverse transcription-coupled quantitative polymerase chain reaction. The eicosanoid synthesis was examined using a radiolabeled arachidonic acid substrate and enzyme-linked immunosorbent assay. We confirmed the absorption of sigma-1 receptor ligands and confirmed that the ligands were not present during the ex vivo studies, so their acute effect could be excluded. We detected no changes in either sigma-1 receptor or cyclooxygenase mRNA levels in the platelets. Nevertheless, (S)-L1 and NE-100 increased the quantity of cyclooxygenases there. Both platelet and aortic eicosanoid synthesis was modified by the ligands, although in different ways. The effect of the new sigma-1 receptor ligand, (S)-L1, was similar to that of PRE-084 in most of the parameters studied but was found to be more potent. Our results suggest that sigma-1 receptor ligands may act at multiple points in arachidonic acid metabolism and play an important role in the control of the microcirculation by modulating the eicosanoid synthesis of the platelets and vessels.
Palotas M, etal., Int J Neuropsychopharmacol. 2004 Dec;7(4):401-13. Epub 2004 Jul 26.
The effect of antidepressants is the culmination of a series of molecular actions occurring in the brain. These events are thought to lead to changes in the expression level of numerous, but as yet unknown genes that result in different cellular functions. In our present study we addressed this issu
e by establishing gene expression profiles of the rat brain after treatment with imipramine and citalopram at therapeutic doses. After 96 h and 4 wk, fronto-temporal cortices from controls and each treated strain were prepared and total RNA was isolated, and assessed using a cDNA microarray system containing 3200 clones. The expression of 6 genes was decreased and 8 were over-expressed by imipramine, whereas 27 were repressed and 7 were up-regulated by citalopram. Members of signal transduction (e.g. phosphatidylinositol transfer protein), structural elements (e.g. tubulin, fibronectin), factors related to protein metabolism in general (e.g. proteasomal subunits, ubiquitin-like proteins, polyadenylation sites), components involved in cell survival (e.g. midkine, stress-inducible protein), and determinants of membrane conductance and ion transport (e.g. vacuolar H+-ATPase), and basics of nuclear functions (e.g. translin, basal transcription factor 3), were some of the genes with altered expression. These data demonstrate that antidepressants interfere with the expression of a large array of genes involved in signalling, survival and protein metabolism. Our results demonstrate for the first time that antidepressants specifically regulate neuronal plasticity through induction of a highly specific transcriptional programme in brain cells.
Kálmán J, etal., Life Sci. 2004 Jul 23;75(10):1195-204.
The inheritance of the apolipoprotein E4 (APOE4) allele has been shown to increase the plasma cholesterol level, but little information is as concerns the association of the APOE genotype and hyperlipidaemia and the activities of two serum enzymes, acetylcholinesterase (AChE) and butyrylcholinestera
se (BChE). Blood samples from 55 type IIb hyperlipidaemic, non-demented patients and 55 age- and sex-matched controls were therefore examined in this pilot study. A significantly increased BChE activity was found in the serum of type IIb hyperlipidaemic patients, but the AChE activity did not differ significantly as compared with that in the control group. The APOE4 allele was significantly overrepresented among the hyperlipidaemic probands, but neither serum cholinesterase activity was affected by the dosage of the APOE4 gene. Our results point to a possible association between an abnormal lipid metabolism and the BChE activity and might have implications as regards the pathomechanism of both Alzheimer's and vascular dementias and the cholinesterase inhibitor therapy of dementing disorders.
The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with alpha-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of alpha-synuclein with beta-amyloid (Abeta) in human brain incl
usions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Abeta oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Abeta with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Abeta(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Abeta(42), alpha-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Abeta was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Abeta can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Abeta and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.
Nicotinamide phosphoribosyltransferase (NAMPT) is a regulator of the intracellular nicotinamide adenine dinucleotide (NAD) pool. NAD is an essential coenzyme involved in cellular redox reactions and is a substrate for NAD-dependent enzymes. In various metabolic disorders and during ageing, levels of
NAD are decreased. Through its NAD-biosynthetic activity, NAMPT influences the activity of NAD-dependent enzymes, thereby regulating cellular metabolism. In addition to its enzymatic function, extracellular NAMPT (eNAMPT) has cytokine-like activity. Abnormal levels of eNAMPT are associated with various metabolic disorders. NAMPT is able to modulate processes involved in the pathogenesis of obesity and related disorders such as nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) by influencing the oxidative stress response, apoptosis, lipid and glucose metabolism, inflammation and insulin resistance. NAMPT also has a crucial role in cancer cell metabolism, is often overexpressed in tumour tissues and is an experimental target for antitumour therapies. In this Review, we discuss current understanding of the functions of NAMPT and highlight progress made in identifying the physiological role of NAMPT and its relevance in various human diseases and conditions, such as obesity, NAFLD, T2DM, cancer and ageing.
Alarmo EL, etal., Genes Chromosomes Cancer. 2016 Mar;55(3):227-36. doi: 10.1002/gcc.22324. Epub 2015 Dec 18.
Bone morphogenetic protein 4 (BMP4) is a remarkably powerful inhibitor of breast cancer cell proliferation, but it is also able to induce breast cancer cell migration in certain cellular contexts. Previous data demonstrate that BMP4 controls the transcription of a variety of protein-coding genes, b
ut not much is known about microRNAs (miRNA) regulated by BMP4. To address this question, miRNA expression profiles following BMP4 treatment were determined in one mammary epithelial and seven breast cancer cell lines using microarrays. While the analysis revealed an extensive variation in differentially expressed miRNA across cell lines, four miRNAs (miR-16-5p, miR-106b-5p, miR-23a-3p, and miR-23b-3p) were commonly induced in a subset of breast cancer cells upon BMP4 treatment. Inhibition of their expression demonstrated an increase in BT-474 cell number, indicating that they possess tumor suppressive properties. However, with the exception of miR-106b-5p, these effects were independent of BMP4 treatment. Scratch assay with miR-16-5p and miR-106b-5p inhibitors on BMP4-treated MDA-MB-231 cells resulted in enhanced cell migration, suggesting that these miRNAs are engaged in BMP4-induced motility. Taken together, we have for the first time characterized the BMP4-induced miRNA expression profiles in breast cancer cell lines, showing that induced miRNAs contribute to the fine-tuning of proliferation and migration phenotypes.
Rubinstein-Taybi syndrome (RSTS) is a rare autosomal dominant congenital disorder (prevalence, 1:125000-720000) characterized by broad thumbs and halluces, facial dysmorphism, psychomotor development delay, skeletal defects, abnormalities in the posterior fossa, and short stature. The purpose of th
is study was to use targeted exome sequencing to identify the genetic cause of RSTS in a 6.5-year-old girl presenting typical features of this condition. Targeted exome sequencing of the patient DNA revealed de novo transition c.1066C>T corresponding to a novel nonsense mutation p.Q356X in the CREB-binding protein gene, CREBBP, whose haploinsufficiency is responsible for 50% to 60% of the RSTS cases. Based on comparing the clinical manifestations of our patient with those of patients carrying similar mutations, we supposed that haploinsufficiency is the possible functional consequence of p.Q356X mutation by creation of a loss-of-function CREBBP allele due to a premature stop codon and RSTS phenotype. Our findings expand the spectrum of mutations associated with this condition.
Meis1 overexpression induces tumorigenicity but its activity is inhibited by Prep1 tumor suppressor. Why does overexpression of Meis1 cause cancer and how does Prep1 inhibit? Tumor profiling and ChIP-sequencing data in a genetically-defined set of cell lines show that: 1) The number of Meis1 and Pre
p1 DNA binding sites increases linearly with their concentration resulting in a strong increase of "extra" target genes. 2) At high concentration, Meis1 DNA target specificity changes such that the most enriched consensus becomes that of the AP-1 regulatory element, whereas the specific OCTA consensus is not enriched because diluted within the many extra binding sites. 3) Prep1 inhibits Meis1 tumorigenesis preventing the binding to many of the "extra" genes containing AP-1 sites. 4) The overexpression of Prep1, but not of Meis1, changes the functional genomic distribution of the binding sites, increasing seven fold the number of its "enhancer" and decreasing its "promoter" targets. 5) A specific Meis1 "oncogenic" and Prep1 "tumor suppressing" signature has been identified selecting from the pool of genes bound by each protein those whose expression was modified uniquely by the "tumor-inducing" Meis1 or tumor-inhibiting Prep1 overexpression. In both signatures, the enriched gene categories are the same and are involved in signal transduction. However, Meis1 targets stimulatory genes while Prep1 targets genes that inhibit the tumorigenic signaling pathways.
Turro E, etal., Sci Transl Med. 2016 Mar 2;8(328):328ra30. doi: 10.1126/scitranslmed.aad7666. Epub 2016 Mar 2.
The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in
SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of alpha-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.
Stritt S, etal., Blood. 2016 Jun 9;127(23):2903-14. doi: 10.1182/blood-2015-10-675629. Epub 2016 Feb 24.
Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We rep
ort the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.
Wunderlich KA, etal., Invest Ophthalmol Vis Sci. 2010 Sep;51(9):4809-20. Epub 2010 Mar 31.
PURPOSE: To examine in rodent models of retinitis pigmentosa (RP) the expression of the neuroprotectants metallothionein-I and -II and of megalin, an endocytic receptor that mediates their transport into neurons. METHODS: Gene and protein expression were analyzed in retinas of rd1 and rds mice and
in those of RCS (Royal College of Surgeons) rats of various ages. Glial cell markers (cellular retinaldehyde binding protein, CRALBP; glial fibrillary acidic protein, GFAP; CD11b; and isolectin B4) were used to establish the identity of the cells. RESULTS: Metallothionein-I and -II gene expression increased with age in normal and degenerating retinas and was significantly greater in the latter. Protein expression, corresponding to metallothionein-I+II, was first observed in rd1 mice in Muller cells at postnatal day (P)12 and in rds mice at P16, coinciding with the onset of GFAP expression in these cells. In RCS rats, the same distribution was observed, but not until P32, long after the onset of GFAP expression. Metallothionein-I+II was observed also in a small number of microglial cells. Megalin was expressed in the nerve fiber layer and in the region of the inner and outer segments in normal animals, but expression in the outer retina was lost with age in degenerating retinas. CONCLUSIONS: Induction of metallothionein-I and -II occurs in the RP models studied and correlates with glial activation. The progressive loss of megalin suggests that transport of metallothionein-I+II into the degenerating photoreceptors (from e.g., Muller cells), could be impaired, potentially limiting the actions of these metallothioneins.
Alfonso J, etal., J Neurosci. 2015 Oct 7;35(40):13659-72. doi: 10.1523/JNEUROSCI.1353-15.2015.
Neuroblast migration is a highly orchestrated process that ensures the proper integration of newborn neurons into complex neuronal circuits. In the postnatal rodent brain, neuroblasts migrate long distances from the subependymal zone of the lateral ventricles to the olfactory bulb (OB) within the ro
stral migratory stream (RMS). They first migrate tangentially in close contact to each other and later radially as single cells until they reach their final destination in the OB. Sphingosine 1-phosphate (S1P) is a bioactive lipid that interacts with cell-surface receptors to exert different cellular responses. Although well studied in other systems and a target for the treatment of multiple sclerosis, little is known about S1P in the postnatal brain. Here, we report that the S1P receptor 1 (S1P1) is expressed in neuroblasts migrating in the RMS. Using in vivo and in vitro gain- and loss-of-function approaches in both wild-type and transgenic mice, we found that the activation of S1P1 by its natural ligand S1P, acting as a paracrine signal, contributes to maintain neuroblasts attached to each other while they migrate in chains within the RMS. Once in the OB, neuroblasts cease to express S1P1, which results in cell detachment and initiation of radial migration, likely via downregulation of NCAM1 and beta1 integrin. Our results reveal a novel physiological function for S1P1 in the postnatal brain, directing the path followed by newborn neurons in the neurogenic niche. SIGNIFICANCE STATEMENT: The function of each neuron is highly determined by the position it occupies within a neuronal circuit. Frequently, newborn neurons must travel long distances from their birthplace to their predetermined final location and, to do so, they use different modes of migration. In this study, we identify the sphingosine 1-phosphate (S1P) receptor 1 (S1P1) as one of the key players that govern the switch from tangential to radial migration of postnatally generated neuroblasts in the olfactory bulb. Of interest is the evidence that the ligand, S1P, is provided by nearby astrocytes. Finally, we also propose adhesion molecules that act downstream of S1P1 and initiate the transition from tangential chain migration to individual radial migration outside of the stream.
Kircher P, etal., Sci Signal. 2015 Nov 10;8(402):ra112. doi: 10.1126/scisignal.aad2959.
Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor (SRF) that promotes the expression of genes associated with cell proliferation, motility, adhesion, and differentiation-processes that also involve dynamic cytoskeletal changes in the cell. MKL1 is inactive when bound to mo
nomeric globular actin (G-actin), but signals that activate the small guanosine triphosphatase RhoA cause actin polymerization and MKL1 dissociation from G-actin. We found a new mechanism of MKL1 activation that is mediated through its binding to filamin A (FLNA), a protein that binds filamentous actin (F-actin). The interaction of FLNA and MKL1 was required for the expression of MKL1 target genes in primary fibroblasts, melanoma, mammary and hepatocellular carcinoma cells. We identified the regions of interaction between MKL1 and FLNA, and cells expressing an MKL1 mutant that was unable to bind FLNA exhibited impaired cell migration and reduced expression of MKL1-SRF target genes. Induction and repression of MKL1-SRF target genes correlated with increased or decreased MKL1-FLNA interaction, respectively. Lysophosphatidic acid-induced RhoA activation in primary human fibroblasts promoted the association of endogenous MKL1 with FLNA, whereas exposure to an actin polymerization inhibitor dissociated MKL1 from FLNA and decreased MKL1-SRF target gene expression in melanoma cells. Thus, FLNA functions as a positive cellular transducer linking actin polymerization to MKL1-SRF activity, counteracting the known repressive complex of MKL1 and monomeric G-actin.
Lentaigne C, etal., Blood. 2019 Dec 5;134(23):2070-2081. doi: 10.1182/blood.2019000782.
To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an asso
ciation between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.
Metallothionein-III is a low molecular weight, heavy-metal binding protein expressed mainly in the central nervous system. First identified as a growth inhibitory factor (GIF) of rat cortical neurons in vitro, it has subsequently been shown to be a member of the metallothionein (MT) gene family and
renamed as MT-III. In this study we have raised polyclonal antibodies in rabbits against recombinant rat MT-III (rMT-III). The sera obtained reacted specifically against recombinant zinc-and cadmium-saturated rMT-III, and did not cross-react with native rat MT-I and MT-II purified from the liver of zinc injected rats. The specificity of the antibody was also demonstrated in immunocytochemical studies by the elimination of the immunostaining by preincubation of the antibody with brain (but not liver) extracts, and by the results obtained in MT-III null mice. The antibody was used to characterize the putative differences between the rat brain MT isoforms, namely MT-I+II and MT-III, in the freeze lesion model of brain damage, and for developing an ELISA for MT-III suitable for brain samples. In the normal rat brain, MT-III was mostly present primarily in astrocytes. However, lectin staining indicated that MT-III immunoreactivity was also present in microglia, monocytes and/or macrophages in the leptomeninges and lying adjacent to major vessels. In freeze lesioned rats, both MT-I+II and MT-III immunoreactivities increased in the ipsilateral cortex. The pattern of MT-III immunoreactivity significantly differed from that of MT-I+II, since the latter was evident in both the vicinity of the lesioned tissue and deeper cortical layers, whereas that of the former was located only in the deeper cortical layers. This suggests different roles for these MT isoforms, and indeed in a new bioassay measuring astrocyte migration in vitro, rMT-III promoted migration to a higher extent than MT-I+II. Thus, MT-III could not only affect neuronal sprouting as previously suggested, but also astrocyte function. Finally, MT-III protein levels of patients with Alzheimer's disease (AD) were, if anything, increased when compared with similarly aged control brains, which was in agreement with the significantly increased MT-III mRNA levels of AD brains.
ER-bound PTP1B is expressed in hippocampal neurons, and accumulates among neurite contacts. PTP1B dephosphorylates ß-catenin in N-cadherin complexes ensuring cell-cell adhesion. Here we show that endogenous PTP1B, as well as expressed GFP-PTP1B, are present in dendritic spines of hippocampal neurons
in culture. GFP-PTP1B overexpression does not affect filopodial density or length. In contrast, impairment of PTP1B function or genetic PTP1B-deficiency leads to increased filopodia-like dendritic spines and a reduction in mushroom-like spines, while spine density is unaffected. These morphological alterations are accompanied by a disorganization of pre- and post-synapses, as judged by decreased clustering of synapsin-1 and PSD-95, and suggest a dynamic synaptic phenotype. Notably, levels of ß-catenin-Tyr-654 phosphorylation increased ∼5-fold in the hippocampus of adult PTP1B(-/-) (KO) mice compared to wild type (WT) mice and this was accompanied by a reduction in the amount of ß-catenin associated with N-cadherin. To determine whether PTP1B-deficiency alters learning and memory, we generated mice lacking PTP1B in the hippocampus and cortex (PTP1B(fl/fl)-Emx1-Cre). PTP1B(fl/fl)-Emx1-Cre mice displayed improved performance in the Barnes maze (decreased time to find and enter target hole), utilized a more efficient strategy (cued), and had better recall compared to WT controls. Our results implicate PTP1B in structural plasticity within the hippocampus, likely through modulation of N-cadherin function by ensuring dephosphorylation of ß-catenin on Tyr-654. Disruption of hippocampal PTP1B function or expression leads to elongation of dendritic filopodia and improved learning and memory, demonstrating an exciting novel role for this phosphatase.
Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typicall
y with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies1-3. Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed4) identified multiple new candidate PID-associated genes, including IVNS1ABP. We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of-and interplay between-novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans.
